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727.Full.Pdf ALLELE SPECIFIC DETERMINANTS OF HOMOTHALLISM IN SACCHAROMYCES LACTISI ALBERTA HERMAN293 AND HERSCHEL ROMAN Department of Genetics, University of Washington, Seattle Received November 29, 1965 N general, fertile yeasts are classified as being either heterothallic or homo- thallic. Heterothallic strains exhibit one of two alternate mating types (usually ’ a! designated as a and or + and -) with diploidization occurring between cells of opposite mating type only (e.g. a with a! and + with -) . In homothallic yeasts, on the other hand, restrictive mating systems are not observed and sister cells quite commonly fuse to form zygotes. The known genetic controls regulating homothallism in yeast are numerous and apparently complex in their action. For example, LEUPOLD(1958) working with the fission yeast Schizosaccharomyces pombe strain liquefaciens found that mating behaviour was controlled by a complex locus. Two heterothallic strains, h+ and h-, were genetically determined by closely linked mutant sites within this complex locus. Homothallic strains, designated H40 and Hgo,could be derived from these heterothallic cultures either through mutation or through recombina- tion within the complex region. In the yeast Saccharomyces chevalieri the pres- ence of a gene D in a single-spore culture committed the culture to homothallism (WINGEand ROBERTS1949). The D gene was independent of the mating locus and was equally effective in the presence of either the a or alleles. Heterothallic cells of S. chevalieri, on the other hand, possessed the recessive d allele and exhibited either one of the alternate mating types a or a. OESER(1962) and HAWTHORNE(1963b) have reported that the D gene produces homothallism by directing mutation of a mating type allele to the alternate form in some of the cells within a culture. Following this mutation, the affected culture becomes homothallic since it consists of a mixture of a and a! sister cells capable of zygote formation. In Saccharomyces cerevisiae homothallism results either from a high mutation rate of one mating type allele to the other (AHMAD1952) or from the action of the complementary genes HM,HM, and HM,HM, (TAKAHASHI,SAITO, and IKEDA1958; TAKAHASHI1958). To compare the nature of the D and HM genes, TAKAHASIand IKEDA( 1959) prepared hybrid Saccharomyces diploids containing both the HM,HM, complement and the D gene of WINGEand ROBERTS(1949). The D gene and the HM genes segregated independently. This paper reports the identification of factors affecting homothallism in ’ This recearch supported by Public Health Service grants 1459 and 5R01 (AT-00328-14) Public Health Service postdoctoral fellowship 5-FZ-GM-13, 112-02 Present address Northern Utilization Reseaich and Development Di\ision, ‘IRS, U S Department of Agriculture, Peoria, Illinois Genetlcs 53: i27-740 Apnl 1966 728 A. HERMAN AND H. ROMAN selected strains of the yeast Saccharomyces Zactis. Two unlinked independent homothallic genes were identified. These factors exert their effect by changing the mating type to the alternative form in a certain percentage of the cells. Moreover, these two genes are allele specific. One locus is effective in changing the mating type from a to (Y, the other in changing a to a. MATERIALS AND METHODS Parental stocks Y14 and Y123 were obtained from L. J. WICKERHAM(strains Y1140 and Y1118 respectively). All isolates used in this study were derived from matings in which these two strains were the original parents. The maintenance of stocks and the procedures used in tetrad dissections and carbohydrase assays were described previously (HERMANand HALVORSON 1963). Cells grown for 24 hours on YM (WICKERHAM1951) were used as mating inocula. Malt extract agar (ME) (WICXWMAN1%1) was used for mating and sporulation. Vegetative growth in Saccharomyces lactis consists essentially of haploid cells. The diploid phase is limited, in the majority of cases, to the zygote formed by the mating of two haploid cells. Usually the zygote forms four haploid ascospores per ascus. Since mating is immediately followed by sporulation, ME serves both as a mating and sporulating medium. In both homothallic cultures and hetero- thallic mating mixtures, zygote formation was confined exclusively to cells grown on ME. Cells inoculated onto ME do not immediately mate but undergo a period of multiplication (8 to 10 hr) before the first competent cells appear in the population. Whether the cells from the original inoculum ever become competent or whether the mating response is confined exclusively to cells formed during growth on ME is not known. Following the 8 to 10 hr period of vegetative growth the proportion of cells capable of producing zygotes gradually increases until a maximum degree of competency is attained after 3 to 5 days incubation. Homothallic and heterothallic cultures are differentiated on the basis of differences in their mating response during growth on ME at 28°C. Homothallic cells form zygotes when grown alone on ME. Heterothallic strains exhibit no mating response under these same conditions but produce zygotes after inoculation in mixed culture with one of the two tester strains, either a or a. As will be seen later, most of the homothallic strains were weakly homothallic, i.e., less than 25% of the cells in a population formed zygotes. During this study it was necessary to determine the homothallic potentialities of several segregants. This was done by counting the number of zygotes formed per 100 vegetative cells after 3 to 5 days incubation at 28°C and is referred to as the percent homothallism of a culture. In addition to determining the percent homothallism, several cultures were also examined for the presence of cells possessing a or a mating potentialities. For this determination, standard inocula of the strain to be tested were mixed and plated with equal volumes of the a or a testers on ME. After 4 days incubation at Doc,the ratio of zygotes to vegetative cells was determined in a sample of each cell mixture and compared with the ratio in the homothallic strain grown by itself. The rationale of this procedure was as follows: In the presence of an excess of a tester cells, each cell in the homothallic population possessing CY characteristics should mate. Similarly, a challenge with an excess of a tester cells should initiate zygote formation by each cell with a qualities. Thus any marked increase in the zygote : vegetative-cell ratio in mixed matings com- pared to homothallic control implied the presence of cells possessing a or a mating potentialities in the homothallic culture. Usually 400 to 500 cells were counted per sample. Larger numbers were counted when the ratio of zygotes to vegetative cells was less than 1% in the homothallic strain. Generally the homothallic segregants contained an excess of competent a or CY cells. Those possessing more a cells were designated as a-associated homothallic strains; those with more a cells as @-associatedhomothallic cultures. Tetrads exhibiting normal segregation patterns for adenine, histidine, leucine and/or trypto- phan nutritional markers as well as for the carbohydrases described previously (HERNIANand HALVORSON1963) were used in this study. With the exception of one adenine requiring mutation, none of the markers used was closely linked to the mating locus. The adenine mutant controlled HOMOTHALLISM IN YEAST 729 the production of a red pigment and was linked to the mating locus with a recombination fre- quency of about 4%. Each stock is identified by a tetrad number and a spore-culture letter A through D. The crosses and tetrads derived from each hybridization which were used in this study are listed in Table 1. In all the hybridizations considered in this paper, the complete genotype relating to mating type is listed. This is followed, in parentheses, by the stock number of the respective parents used in each cross. RESULTS The parental mating types: Parental cultures Y14 and Y123 mated and sporu- lated when they were mixed and grown on ME plates. Neither strain showed a mating response when grown alone on ME. They were, therefore, identified as heterothallic cultures. Strain Y14 was designated as the a mating type, strain Y123 as the a: mating type. Evidence for homothallism: When the mating type of progeny derived from the parental cross a X a: (Y14 x Y123) was determined, both homothallic and heterothallic segregants were identified. Segregation ratios for homothallism: heterothallism included 3:l (3 tetrads), 2:2 (50 tetrads), 1:3 (77 tetrads), and 0:4 (26 tetrads). The 3 homothallic: 1 heterothallic ratio indicated that the hybrid was heterozygous for more than one locus for homothallism while the 0 homo- thallic:4 heterothallic ratio implied that the homothallic condition required spe- cific combinations of the homothallic factors and the mating type alleles. In the 26 tetrads in which all of the progeny were heterothallic, segregation of a and a: mating types could be followed readily. The ratio of a:a: was always 2:2 and indicated that a and a: were segregating as alleles of the mating locus. It was inferred that a and N segregated in this manner in all of the tetrads even though TABLE 1 Hybridiurtions and tetrads used in this investigation ___ Hybridization Tetrad number ah,H, x aH,h, (Y14 x Y123) 155 157 31 1 338 599 729 1009 cuh,H, (729B*) 1216 aH,h, (155C') 25 1 &,ha (338C') 95 3 cuH,h, (251A*) 1254 1256 aH,h, (1256C') 1290 aH,h, X ah,H, (251A' x Y14) 1255 1257 1258 * IIomothallic. 730 A. HERMAN AND H. ROMAN this segregation was obscured in many tetrads by the presence of the homothallic condition. TOaccount for the above observations, the following hypothesis is proposed: In addition to the mating type alleles a and a, there are two nonallelic genes for homothallism, Ha and Ha. The combinations CUH, (either ahaH, or aHaHa) and da(either &aha or aHaHa)result in homothallism.
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